Petroleum derived fuels have been the primary source of energy for over a hundred years. Petroleum is formed over millions of years in nature and is a non-renewable source of energy. A significant amount of research in biofuels has been ongoing for decades. Within this field, ethanol has been studied intensively as a gasoline substitute. However, the efficiency of ethanol as a fuel remains debatable. (Pimentel, Natural Resources Research (2005) 14:65; Farrell et al., Science (2006), 311:506).
The present invention provides methods for the biosynthesis of branched 5-carbon alcohols (branched-C5 alcohols) from the isoprenoid precursors isopentyl pyrophosphate (IPP) or dimethylallyl diphosphate (DMAPP).
Isoprenoids are compounds derived from the five-carbon molecule, IPP. Investigators have identified over 29,000 individual isoprenoid compounds, with new ones continuously being discovered. Isoprenoids are often isolated from natural products, such as plants and microorganisms, which use isopentenyl pyrophosphate as a basic building block to form relatively complex structures. Vital to living organisms, isoprenoids serve to maintain cellular fluidity and electron transport, as well as function as natural pesticides, to name just a few of their roles in vivo. Furthermore, the pharmaceutical and chemical communities use isoprenoids as pharmaceuticals, nutriceuticals, flavoring agents, and agricultural pest control agents. Given their importance in biological systems and usefulness in a broad range of applications, isoprenoids have been the focus of much attention by scientists.
Conventional means for producing isoprenoids include extraction from biological materials (e.g., plants, microbes, and animals) and partial or total organic synthesis in the laboratory. Such means, however, have generally been unsatisfactory as they involve the use of toxic solvents and provide a low yield of the desired isoprenoid. Recently, researchers have looked to the biosynthetic production of isoprenoids. U.S. Pat. No. 6,291,745 describes the production of limonene and other metabolites in plants. U.S. Pat. No. 6,190,895 describes nucleic acid sequences that code for the expression of 1-deoxyxylulose-5-phosphate synthase, an enzyme used in one biological pathway for the synthesis of isopentenyl pyrophosphate. U.S. Pat. No. 7,172,886 describes the cloning of genes for a mevalonate-isoprenoid pathway and synthesizing an isoprenoid or an isoprenoid precursor via the mevalonate pathway in a host cell. U.S. Pat. No. 7,183,089 describes a method for enhancing production of isoprenoid compounds in a host cell by modulating the level of hydroxymethylglutaryl-CoA (HMG-CoA) in the cell, such that the level of HMG-CoA is not toxic to the cell and does not substantially inhibit cell growth.
U.S. Pat. Nos. 5,460,949; 6,531,303; and 6,689,593; U.S. Pat. Pub. Nos. 2003/0148479; 2004/0029239; 2004/005678; 2004/0063182; 2004/0072323; 2004/0077039; 2004/0110259; and 2004/0194162; Martin et al. (2003) Nat. Biotech. 21(7):796-802; Polakowski et al. (1998) Appl. Microbial. Biotechnol. 49:67-71; Wilding et al. (2000) J. Bacterial. 182(15):4319-27; Donald et al. (1997) Appl. Env. Microbial. 63:3341-3344; Jackson et al. (2003) Organ. Lett. 5:1629-1632; Hamano et al. (2001) Biosci. Biotechnol. Biochem. 65:1627-1635; Kuzuyama (2004) Biosci. Biotechnol. Biochem. 68(4):931-934; Kazuhiko (2004) Biotechnol. Lett. 26:1487-1491; Brock et al. (2004) Eur. J. Biochem. 271:3227-3241; Choi et al. (1999) Appl. Environ. Microbiol. 65:4363-4368; Parke et al. (2004) Appl. Environ. Microbiol. 70:2974-2983; Subrahmanyam ct al. (1998) J. Bacteriol. 180:4596-4602; Murli et al. (2003) J. Ind Microbiol. Biotechnol. 30:560-509. These references are hereby incorporated in their entireties by reference.